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. 2013 Nov 5;8(11):e80319. doi: 10.1371/journal.pone.0080319

Figure 4. Chemical chaperones partially restore Bmpr2ΔEx2 mutant product expression at the cell surface.

Figure 4

A, Cell surface expression of Bmpr2ΔEx2 in Bmpr2 ΔEx2/+ ciPECs treated with 4-PBA. Bmpr2 ΔEx2/+ ciPECs were treated for 48 hours with 100µM, 250µM, 500µM or 1mM of 4-PBA. Monolayers were then labeled with membrane impermeable biotin and biotinylated cell surface proteins pulled-down with streptavidin agarose beads. Western Blot was performed with Clone 18 anti-BMPR2 antibody. The 150 kDa wild type Bmpr2 product was detected in the streptavidin pull-down in control and Bmpr2 ΔEx2/+ ciPECs, but the 130 kDa Bmpr2ΔEx2 mutant product was not detected. After treating with the chemical chaperone 4-PBA, the 130kDa Bmpr2ΔEx2 mutant product was detected and there was increased expression of the wild type Bmpr2 product in the streptavidin pull-down. Numbers shown below the upper panel indicate the ratio of the 130 kDa Bmpr2ΔEx2 band before and after treatment with 4-PBA. Lower panel, expression of wild type Bmpr2 and Bmpr2ΔEx2 in ciPEC cell lysates with 4-PBA treatment. B, Quantification of wild type Bmpr2 and Bmpr2ΔEx2 band densities after 4-PBA treatment relative to untreated controls from three independent experiments, standard error is indicated. C, Cell surface expression of Bmpr2ΔEx2 in Bmpr2 ΔEx2/+ ciPECs treated with TUDCA. Wild type and Bmpr2 ΔEx2/+ ciPECs were treated for 5 hours with 50µM 100µM, 250µM or 500µM of TUDCA. Streptavidin pull-down shows that the 130kDa Bmpr2ΔEx2 mutant product was partially restored at the cell surface and there was a slight increase in wild type Bmpr2 with TUDCA treatment (1.4 fold increase with 500µM TUDCA versus untreated cells). Numbers shown below the upper panel indicate the ratio of the 130 kDa Bmpr2ΔEx2 band before and after treatment with TUDCA Lower panel, expression of wild type Bmpr2 and Bmpr2ΔEx2 in ciPEC cell lysates with TUDCA treatment. D, Quantification of wild type Bmpr2 and Bmpr2ΔEx2 band densities after TUDCA treatment relative to untreated controls from three independent experiments.